Enzyme-macromolecule conjugates are typically used for the detection and determination of substances present in very low quantities; for example, nanogram quantities of substances in biological fluids, such as urine and serum. A wide variety of enzymes may be used to form the conjugate, but the enzymes selected are often those enzymes which can be detected with great sensitivity. The macromolecular portion of the conjugate presents a wide variety of amino-containing compounds, including, but not limited to, nucleic acids, proteins, hormones, antigens and allergens, which are characterized in containing amino groups.
Enzymes conjugates are prepared in a conjugation reaction with a polyfunctional coupling reagent which links the enzyme and macromolecule together by reaction with one or more of the reactive groups in the reactants. In the preparation of enzyme conjugates, it is most desirable to produce enzyme conjugates of high stability, high specificity and good reproducibility.
In some coupling reactions to prepare enzyme conjugates, it has been suggested to employ a conditioner compound, such as a polyamine, to improve specificity of the enzyme conjugate, with resulting improvement in the detection method due to low signal-to-noise ratio during detection in the immunochemical test. The preparation of enzyme conjugates, employing prior-art coupling reagents with conditioners, is described in U.S. Pat. No. 4,002,532, issued Jan. 1, 1977, hereby incorporated by reference.
A new coupling agent has been described for the preparation of an enzyme-coupled insulin conjugate for use in the immunoassay of insulin. The coupling agent is meta-maleimidobenzoyl N-hydroxysuccinimide ester (MBS), a bifunctional reagent which acylates the amino groups of the insulin by reaction with the N-hydroxysuccinimide ester group and by forming thioester bonds with the enzyme by addition of the thiol groups to the maleimide group. This coupling agent has been employed in preparing an enzymatically active and immuno-reactive B-D-galactosidase-MBS-insulin conjugate (see J.Biochem., 79, 233-236 (1976), "Enzyme Coupled Immunoassay of Insulin Using a Novel Coupling Reagent", Kitagawa, T and Aikawa, T, hereby incorporated by reference). Although the MBS coupling reagent is satisfactory in some respects, it is desirable to obtain enzyme conjugates of greater stability and greater specificity and sensitivity.